AUMantagomir™ ASO Protocol
Comprehensive in vitro protocol for miRNA inhibition using self-delivering antisense oligonucleotides
Introduction
This protocol provides detailed instructions for using AUMantagomir™ self-delivering antisense oligonucleotides (sdASO™) in mammalian cell culture. AUMantagomir™ ASOs enable efficient miRNA inhibition without the need for transfection reagents, making them ideal for a wide range of cell types, including difficult-to-transfect cells.
Protocol Overview
AUMantagomir™ sdASO™ delivery is a simple, three-step process:
- Plate cells at optimal density (30-50% confluency)
- Add AUMantagomir™ sdASO™ directly to culture medium
- Incubate and analyze results (typically 24-72 hours)
This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.
- No Transfection Required: Simply add to media - no lipofection, electroporation, or viral vectors needed
- Universal Cell Compatibility: Works in difficult-to-transfect cell types including primary cells, neurons, and immune cells
- High Specificity: Precisely targets specific miRNAs with minimal off-target effects
- Low Toxicity: Maintains cell viability without transfection reagent-associated toxicity
Materials and Reagents
Required Items
- AUMantagomir™ sdASO™ (lyophilized or stock solution)
- Appropriate cell culture medium
- Culture plates or vessels
- Mammalian cells of interest
- Sterile nuclease-free water or buffer (for ASO resuspension)
- Microcentrifuge tubes (for aliquoting ASO stock)
- Standard cell culture equipment:
- Sterile pipettes and tips
- Cell culture hood
- Humidified cell culture incubator
- Centrifuge
Detailed Protocol
Cell Preparation
Plate cells in their optimum growth medium at appropriate density for your cell type.
For adherent cells: Plate cells the day before treatment at 30-50% confluency (or at densities optimized for your specific cell type and assay endpoint). Allow cells to adhere overnight.
For suspension cells: Prepare cells at appropriate density shortly before treatment with AUMantagomir™ sdASO™.
Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay. As a general guideline, aim for 30-50% confluency at the time of ASO treatment.
When using 96-well plates, approximately 0.5 × 105 cells per well is recommended for cells similar to HeLa in size. Scale accordingly for different plate formats (see reference table below).
AUMantagomir™ sdASO™ Stock Preparation
Prepare AUMantagomir™ sdASO™ stock solution by reconstituting lyophilized ASOs at the desired concentration. If you already have a stock solution prepared, skip to Step 3.
Resuspend lyophilized AUMantagomir™ sdASO™ using the appropriate volume of sterile nuclease-free water or buffer to achieve the desired stock concentration (typically 100 μM).
Pipette the solution up and down 3-5 times while avoiding the introduction of bubbles.
Let the vial sit at room temperature for 5-10 minutes to ensure complete resuspension.
Centrifuge for 30-45 seconds to collect the solution at the bottom of the tube.
Prepare several aliquots of the stock solution to avoid multiple freeze-thaw cycles.
To avoid degradation, minimize freeze-thaw cycles of your ASO stock. It is strongly recommended to make single-use aliquots of your stock solution and store them at -20°C.
AUMantagomir™ sdASO™ Delivery to Cells
Add AUMantagomir™ sdASO™ to the cells at the desired final concentration. The working concentration can vary from 500 nM to 10 μM depending on the cell type and target miRNA.
For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMantagomir™ sdASO™, or add the ASO stock directly to the media overlaying the cells. Mix gently.
For suspension cells: Either pellet the cells by low-speed centrifugation and gently resuspend the cell pellet in media containing AUMantagomir™ sdASO™, or add the ASO stock directly to the media containing the cells. Mix gently.
It is highly recommended to perform a dose response using 2-3 working concentrations (e.g., 500 nM, 1 μM, and 5 μM) to determine the optimal concentration for your specific miRNA target.
For highly abundant miRNAs or certain cell types, higher concentrations (up to 10-20 μM) may be required.
The optimal ASO concentration may vary depending on the target miRNA abundance, cell type, and assay timing. For most applications, 1-5 μM provides an excellent balance of efficacy and economy.
Incubation and Analysis
Incubate cells with AUMantagomir™ sdASO™ and analyze miRNA inhibition at appropriate time points.
Return cells to the incubator and maintain under standard culture conditions.
Analyze AUMantagomir™ sdASO™-treated cells after the desired time point, typically 24-72 hours post-treatment.
miRNA inhibition can be assessed by:
- Measuring target miRNA levels (qRT-PCR, northern blot)
- Evaluating de-repression of miRNA target genes (qRT-PCR, Western blot)
- Monitoring phenotypic changes associated with miRNA inhibition
- Using luciferase reporter assays containing miRNA binding sites
Since AUMantagomir™ binds to and inhibits miRNAs, you may not always observe a significant reduction in miRNA levels by qRT-PCR. The gold standard for confirming successful miRNA inhibition is measuring the de-repression of known miRNA target genes, as these should increase in expression when the miRNA is inhibited.
Reference Calculations
The table below provides guidelines for AUMantagomir™ sdASO™ amounts and cell numbers for various culture plate formats:
| Cell Culture Plate | 96-well | 24-well | 12-well | 6-well |
|---|---|---|---|---|
| AUMantagomir™ sdASO™ stock (μL)1 | 1 μL | 5 μL | 10 μL | 30 μL |
| AUMantagomir™ sdASO™ used (moles) | 100 pmole | 500 pmole | 1 nmole | 3 nmole |
| Cell culture media (μL) | 100 μL | 500 μL | 1000 μL | 3000 μL |
| Cell number (per well)2 | 0.5 × 105 | 2.5 × 105 | 0.5 × 106 | 1 × 106 |
- The amount of AUMantagomir™ sdASO™ shown yields a final concentration of 1 μM using 100 μM stock.
- The optimal seeding cell density will vary with the cell type, cell size, growth characteristics, and the endpoint of the assay. For this table, HeLa cells at 50% confluency were used at the time of AUMantagomir™ sdASO™ treatment.
Tips and Troubleshooting
Optimization Tips and Best Practices
Dose Optimization
Always perform dose optimization for new cell types or miRNA targets. Start with a concentration range (500 nM, 1 μM, and 5 μM) to determine the optimal balance between inhibition efficiency and economy.
Extended Inhibition
For long-term experiments, miRNA inhibition can be maintained for several days using a single dose. For very fast-growing cells, you may need to add more AUMantagomir™ sdASO™ to the cell culture every 48-72 hours to maintain inhibition.
Appropriate Controls
Include a non-targeting control AUMantagomir™ sdASO™ in your experiments to distinguish specific effects of miRNA inhibition from any potential non-specific effects of the ASO chemistry.
Target Validation
Confirm successful miRNA inhibition by measuring the expression of known miRNA target genes. An effective AUMantagomir™ treatment should result in increased expression of genes normally repressed by the target miRNA.
Troubleshooting Common Issues
Low Inhibition Efficiency
- Increase concentration: Try higher concentrations (up to 20 μM) for highly abundant miRNAs or challenging cell types.
- Extend incubation time: Some miRNA inhibition effects may take longer to manifest (48-96 hours).
- Check miRNA expression: Confirm that your target miRNA is expressed in your cell model under your experimental conditions.
- Try an alternative readout: If you don't see a reduction in miRNA levels by qRT-PCR, look for de-repression of known target genes or use a reporter assay.
No Observable Phenotype
- Verify inhibition: Confirm that the miRNA is effectively inhibited by measuring target gene de-repression.
- Extend observation time: Some phenotypic effects may take time to develop after miRNA inhibition.
- Consider redundancy: Other miRNAs from the same family may compensate for the inhibited miRNA. Consider using multiple AUMantagomir™ sdASO™ to target related miRNAs simultaneously.
- Check experimental conditions: The phenotypic effect of miRNA inhibition might only be observable under specific conditions (e.g., stress, differentiation, or stimulation).
Cell Toxicity
- Reduce concentration: If toxicity is observed, try lower concentrations (500 nM - 1 μM).
- Check cell density: Ensure cells are at optimal density (30-50% confluency) at the time of treatment.
- Consider miRNA function: The miRNA might have an essential role in cell survival or proliferation, and its inhibition could naturally cause growth arrest or apoptosis.
Storage and Additional Information
Storage Conditions
- AUMantagomir™ sdASO™ are shipped in lyophilized form. Upon arrival, store at -20°C.
- Resuspended AUMantagomir™ sdASO™ should be stored in aliquots at -20°C to avoid multiple freeze-thaw cycles.
- For short-term storage (up to 1 week), resuspended ASOs can be kept at 4°C.
Additional Notes
- AUMantagomir™ sdASO™ are compatible with standard cell culture media, including those containing serum.
- No pre-treatment or media change is required before adding AUMantagomir™ sdASO™ to cells.
- AUMantagomir™ sdASO™ can be fluorescently labeled to monitor cellular uptake.
- For phenotypic assays, the timing should be optimized based on both the inhibition kinetics and the turnover of proteins regulated by the target miRNA.
AUMantagomir™ sdASO™ are for research use only. Not for use in diagnostic or therapeutic procedures.
There are several methods to confirm successful miRNA inhibition:
- Direct miRNA measurement: qRT-PCR or northern blotting. Note that bound AUMantagomir™ may interfere with miRNA detection.
- Target de-repression: Measure increased expression of known miRNA target genes by qRT-PCR or Western blot.
- Reporter assays: Use luciferase reporters containing miRNA binding sites - effective inhibition will increase reporter signal.
- Functional readouts: Assays specific to the biological function of the target miRNA.
Ready to Explore miRNA Functions?
Our scientific team is available to help you design optimal AUMantagomir™ sdASO™ for your specific miRNA target and application. Contact us for personalized support or to request a quote.
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